Applying ABI 7000 Prism System - Determination of melting temperature (Tm) of genomic DNA (Nov/21/2005 )
I would like to ask you about using ABI 7000 RT/Q PCR to determine Tm of bacterial genomic DNA.
As designated, this system is applied together with CYBER Green or TaqMan kits as well as primers for targeted sequences to detect/quantify fluorescent PCR. In these cases, with the available calculation protocols, the amplified products are known of Tm, number of copy,...easily.
However, according to few papers, from methodological point of view, thermal profile of genomic DNA is needed to determine G+C% content using ABI system (5700 or later version like 7000).
I have applied exact procedures in the mentioned papers, from DNA preparation, CYBR Green I combination, to setup thermal cycle, fluorescent detector... but could not got expected results. The troubles are following:
1) If the thermal set is a single cycle of 60 oC-15s => 98 oC-20 min: machine didn't work
2) If the thermal set is 20 cycles of temperature above, very scattered/massy signal of fluorescence derivative were resulted
3) If none of 2 DNA diassociation protocols were used, and thermal set as the 1) case, ABI 7000 worked but at the end, no data was collected!
4) If a thermal set of
60 oC - 1 min (1 cycle)
60 oC - 30 s => 98 oC - 2 min (20-40 cycles)
98 oC - 30 s => 60 oC (1 cycle)
Data were collected and analysed but with several peaks of fuorescent derivatives coressponding to several Tm.
I don't know what the reason is. Please tell me how to correct if you can.
can you explain to me exactly what your template is?
I'm having a hard time with this one...are you putting a genomic prep into a melting-curve analysis run on the 7000?
I would assume & expect multiple peaks; it is pretty much impossible to have no fragments in a genomic dna prep, not to mention extrachromosomal stuff like plasmids
now, you did try to run a dissociation analysis, but no data was collected? that's bizarre that it didn't save your data. did you watch any of the run? did it look OK while it was going?
Thank you aimikins!
I used extractedly genomic DNA with size >= 20 KB.
DNA concentration was varied from 10ng/microliter to 100 ng/microliter; CYBR Green was 4000-fold dilution.
Yes, what I need is the Tm in the melting curve scanned and analyzed by the machine automatically.
I am sue that no problem with the DNA I used such as plasmid, RNA, or protein contamination
The program was run normally but seemed not stoped (Idle). I think this is the reason no data was collected!
Do you have any idea?
I don't know why it didn't save your data. that happened with ours once, and the problem was some corrupted files making the software cranky. we had a tech support service call and the problem was fixed.